Techniques of

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Chapter 20 Techniques of Molecular Biology

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foreword

• molecular biology means the understanding
  organism from biology. It is just a tool ,but ,it
  is the most import tool for current research
• The methods depend upon, and were developed
  from, an understanding of the properties of
  biological macromolecules themselves.

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?.gain identification
?.Sequencing structural analysis
?.functional analysis
?.informational integration
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?.gain identification
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gain

• a). Isolation of special genes
• b). Gain and purification of proteins
• c). RNA separation

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a). Isolation of special genes

• Techniques DNA cloning
• PCR
• A golden era of molecular biology was launched
  once it became possible to isolate specific DNA
  segments representing individual genes .
• ----Molecular Biology of Genes

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DNA Cloning

• The ability to construct recombinant DNA
  molecules and maintain them in cells is called
  DNA Cloning.
• ----Molecular
  Biology of Genes
• Topically involves a vector the most common one
  is a plasmid.

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Characteristics of vector DNAs

• 1.an origin of replication
• 2.a selectable marker
• 3.sigle sites for one or more restriction
  enzymes.

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Construction of a genomic DNA library

• Genomic DNA and vector DNA, digested with the
  same restriction enzyme, are incubated together
  with ligase
• The resulting pool or library of hybrid vectors
  is then introduced into E. coli, and the cells
  are plated onto a filter placed over agar medium.
• The filter is removed from the plate and prepared
  for hybridization.

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Construction of a cDNA library

• Isolate mRNA
• use reverse transcriptase to synthesize
  complementary DNA strand from mRNA, then use DNA
  Pol I to synthesize double stranded DNA. Clone
  these cDNAs into appropriate vector (usually
  plasmid or phage)
• Use Oligo dT primer to hybridize to polyA tail of
  mRNA. Primer used by reverse transcriptase for
  extension.
• Reverse transcriptase is a DNA polymerase which
  uses RNA as a template to synthesize
  complementary DNA. Cloned from RNA viruses.

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Construction of a cDNA library
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Difference between of a genomic DNA library and a
cDNA library
properties
genomic DNA library Contain both introns and regulatory sequences
cDNA library Contain neither introns nor regulatory sequences
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PCR

• PCR polymerase chain reaction .A powerful
  method for amplifying particular srgments of DNA
  ,which is carried out entirely biochemically in
  vitro.
• Elements
• 1.Template
• 2.The four nucleotides ---dNTPs
• 3.The enzyme DNA polymerase
• 4. A primer.

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PCR
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Typical steps of PCR

• Denaturation at 94? the double strand melts
  open to single stranded DNA, all enzymatic
  reactions stop .
• Annealing at 54? The more stable bonds last a
  little bit longer (primers that fit exactly) and
  on that little piece of double stranded DNA
  (template and primer), the polymerase can attach
  and starts copying the template.
• Extension at 72? This is the ideal working
  temperature for the polymerase. The bases
  (complementary to the template) are coupled to
  the primer on the 3' side (the polymerase adds
  dNTP's from 5' to 3', reading the template from
  3' to 5' side, bases are added complementary to
  the template)

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Something else Site-directed mutagenesis

• Site-directed mutagenesis A custom-designed
  oligonucleotide harboring a mismatch to a segment
  of cloned DNA can be used to create a directed
  mutation in that cloned DNA . A synthetic DNA
  fragment is used as a tool for changing one
  particular code word in the DNA molecule. This
  reprogrammed DNA molecule can direct the
  synthesis of a protein with an exchanged amino
  acid.

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b). Gain and purification of proteins

• Techniques Electrophoresis
• Chromatography
• Basis
• To purify proteins we make use of their inherent
  similarities and differences.
• Protein similarity is used to purify them away
  from the other non-protein contaminants.
• Differences are used to purify one protein from
  another. Proteins vary from each other in size,
  shape, charge, hydrophobicity, solubility, and
  biological activity.

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chromatography

• In this approach, protein fractions are passed
  though glass columns filled with appropriated
  modified small acrylamide or agarose beads.
• There are various ways columns can be used to
  separate proteins according to their
  characteristics.
• Ion exchange chromatography, gel filtration
  chromatography, Affinity Chromatography ,and so on

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fig 1 principle of Ion exchange chromatography
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Fig 2 Principle of gel filtration
chromatography
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Fig 3 Principle of Affinity Chromatography
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antibodies visualize electrophoretically-separated
proteins.

• The electrophoretically separated proteins are
  transferred to a filter. And this filter is then
  incubate in a solution of an antibody to our
  interested protein. Finally, a chromogenic enzyme
  is used to visualized the filter-bound antibody

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identification
Substance methods
DNA, RNA gel electrophoresis, hybridization
Protein Stain Electrophoresis
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DNA gel electrophoresis
Linear DNA molecules migrate through the gel
toward the positive pole with different rates
according to different size
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pulsed-field gel electrophoresis

• Pulsed-field gel electrophoresis forlong DNAs (up
  to severalMb in length).

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Restriction Endonuleases Cleaves DNA Molecules at
Particular Sites
Recognition sequences and cut sites of various
endonucleases
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Southern blot

• 1. DNA fragments, generated by digestion of a
  DNA molecule by a restriction enzyme, are run out
  on an agarose gel.
• 2. Once stained, a pattern of fragments is seen.
• 3.When transferred to a filter and probed with a
  DNA fragment homologous to just one sequence in
  the digested molecule, a single band is seen,
  corresponding to the position on the gel of the
  fragment containing that sequence.

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Varieties of blots
Blot type Target Probe Applications
Southern DNA DNA or RNA mapping genomic clonesestimating gene numbers
Northern RNA DNA or RNA RNA sizes, abundance,and expression
Western Protein Antibodies protein size, abundance
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?.Sequencing structural analysis
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Outline of this part
Substance Sequencing methods Structural analysis
DNA chain-termination shotgun , sometimes use special strategy No variety
Protein Edman degradation MS/MS Various structure
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Sequencing of DNA and genome

• Techniques
• ?. DNA molecules (radioactively labeled at 5
  termini) are subjected to 4 regiments to be
  broken preferentially at Gs, Cs, Ts, As,
  separately.
• ?. chain-termination method.
• The second one is more current .

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The absence of 3-hydroxyl lead to the
inefficiency of the nucleophilic attack on the
next incoming substrate molecule
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Techniques ?. chain-termination method
DNA molecules (radioactively labeled at 5
termini) are subjected to 4 regiments to be
broken preferentially at Gs, Cs, Ts, As,
separately.
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Techniques ?. chain-termination method
4 systems with dNTP ddGTP, dNTP ddATP d NTP
ddCTP, d NTP ddTTP separately. And even ,we can
read the sequencing gel to get the sequence of
the DNA
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fluorescent chain-terminating nucleotides
With the method of fluorescent chain-terminating
nucleotides, we can carry out high throughput
sequence
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Sequencing a genome

• Question chain-termination method is powerful
  ,however, it can only sequence a fragment of
  800bp as most at time.
• Solution the shotgun sequencing ,

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the shotgun sequencing
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The shortgun strategy permits a partial assembly
of large genome sequence

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read containing identical sequences are assumed
to overlap and are joined to form larger contigs
.And paired-end reading leads to form scaffold .
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Structural analysis of DNA

• The two strands twisting around each other in
  the form of a double helix.
• base pairs adenine-thymine
• guanine-cytosine

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Base pairs
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Schematic model Space-filling model
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Importance

• It is the reveal of the structure of DNA that
  bring the golden era of molecular biology
• It reveal how DNA carries genetic messages and
  how DNA reply .
  

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Sequencing of protein

• Techniques
• 1. Edman degradation
• 2. MS/MS

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Edman degradation
The N-terminal residue is labeled and can be
removed without hydrolyzing the rest of the
peptide. Thus, in each round, one residue is
identified, and that residue represents the next
one in the sequence of the peptide.
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Tandem mass spectrometry
Material travels through the instrument in a
manner that is sensitive to its mass/charge
ratio. It is a method with great accuracy.
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Structural analysis of protein

• Techniques
• 1.NMR
• 2.X-ray diffraction
• 3.Crystalization.

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3-D structure
The polypeptide exit tunnel in 50s subunits.
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?.functional analysis
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functional analysis

• functional analysis of DNA and protein have
  analogical basic method absence and reversion.
• In DNA ,it means the analysis of mutant .
• In protein, it infer to reduce or increase
  quantity ,then observe the change .

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Mutation reveal the function of protein

• Research the disease in human and mutation in
  other organisms.

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Structure determines function

• Function is based on and related with structure
  .
• We also compare and predict according to
  general rule . DNA or proteins which have the
  similar structure or sequence is predicted to
  have similar function.

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Structure imply function and mechanism
High resolution structure of the RuvA-DNA complex
and schematic model of the RuvAB complex bound to
Holliday junction DNA.
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Reporter gene

• Reporter gene is a powerful tool in the
  research in vivo .
• it can tell us more informations .

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?.informational integration
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Revert to the all and the one

• There are some reasons
• 1. molecular are interactional with each other
• 2.compare may give us important hint and
  essential information
• 3.learning the part or molecular is a essential
  way to understand the integer. However ,it is
  only a method ,not the primary and ultima aim.

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informational integration

• Large scale analysis
• bioinformatics
• Proteomics
• Signal transduction pathway
• Interaction between proteins and DNAs .

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The two hybrid Assay
The two hybrid Assay is used to identify proteins
interacting with each other.
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bioinformatics

• The computational methods ,which are used to
  assemble complex genomes and identify both
  protein coding genes associated regulatory DNAs.
• Considerable efforts focus on comparing the
  genetic content of different genomes , and
  thereby determine the basis for organismal
  diversity.

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Comparative genome analysis
An example Comparison of a 34kb region of the
mouse and human genomes
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Proteomics

• Proteomics is concerned with the identification
  of the full set of proteins produced by a cell or
  a tissue under a particular by a particular set
  of conditions.

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Proteomics

• 1. 2-D gel electrophoresis for protein
  separation.
• 2. MS for the precise determination of a
  protein.
• 3. Bioinformatics technology.

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2-D gel electrophoresis
High throughout analysis make proteomics available
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May you enjoy it ! Good luck!
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Thank you !
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